Bcl-xL est un oncogène surexprimé dans plusieurs types de cancers et qui joue un rôle important dans la survie cellulaire en régulant deux processus: l'apoptose et l'autophagie. Récemment, nous avons identifié l'existence d'une nouvelle forme de Bcl-xL qui subit une simple déamidation sur le résidu Asn52. Cette forme monodéamidée est exprimée en conditions contrôles et apparaît spontanément in vitro et in vivo. La déamidation de Bcl-xL produit un mélange de protéines contenant en position 52 soit un résidu Asp, soit un résidu isoAsp. L'objectif de cette thèse est de caractériser les fonctions de ces deux espèces protéiques, et de déterminer comment la monodéamidation de Bcl-xL modifie les fonctions de survie de cet oncogène. Nous avons montré que le mutant déamido-mimétique Bcl-xL N52DN66A conserve la même fonction anti-apoptotique que Bcl-xL native, mais présente une activité autophagique plus grande, et des propriétés oncogéniques et tumorigéniques altérées in vitro, ex vivo et in vivo. Nous avons étudié certains des mécanismes impliqués dans la régulation de l'autophagie et les propriétés oncogéniques comme la voie mTor, les voies de signalisation médiées par l'oncogène Ras, ainsi que l'activité métabolique et l'état souche des cellules. D'autre part, nous avons aussi développé des tests in vitro pour analyser les interactions établies par les formes déamidées de Bcl-xL comportant un isoAsp. L'ensemble de nos données permet de suggérer une régulation des fonctions de Bcl-xL par des mécanismes indépendants de l'apoptose, et renforce l'importance d'explorer les fonctions non apoptotiques de cette protéine pour mettre en évidence sa capacité à promouvoir la survie cellulaire et entraîner la progression du cancer. / Bcl-xL is an oncogene overexpressed in many types of cancer and which promotes cell survival by regulating two cellular processes : apoptosis and autophagy. We have recently identified a new form of this oncogene, which results from the deamidation of Asn52. This monodeamidated form is expressed under control conditions and is ubiquitously found in vitro and in vivo. Bcl-xL monodeamidation produces a mixture of proteins containing either an Asp residue or an IsoAsp residue in position 52. Our goal is to caracterise the functions of both species, and to determine how Bcl-xL monodeamidation modifies the survival functions of this oncogene. We have shown that the deamidomimetic mutant Bcl-xL N52DN66A retains the same anti-apoptotic function as the native protein, but exhibits enhanced autophagic activity and impaired clonogenic and tumorigenic properties in vitro, ex-vivo, and in vivo. We have studied certain of the mechanisms which can be involved in the regulation of autophagy and oncogenic properties of Bcl-xL such as mTor, Ras oncogene signaling pathway, metabolic activity measurement and stemness. We also implement in vitro assays to analyse the interactions established by isoAsp containing forms of Bcl-xL. Altogether our results support the view that deamidation regulates Bcl-xL oncogenic properties through apoptosis-independent mechanisms, and reinforce the importance of deciphering the non apoptotic functions of this protein to tackle its ability to sustain cell survival and drivecancer progression.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A toxoplasmose é uma zoonose amplamente distribuída que afeta mais de um terço da população mundial e de grande importância na saúde pública. A maioria das infecções em humanos por Toxoplasma gondii é assintomática. Entretanto, nos últimos anos, a toxoplasmose tem sido amplamente investigada uma vez que se apresenta como uma das doenças oportunistas que acometem pacientes portadores da Síndrome da Imunodeficiência Adquirida (AIDS) e indivíduos transplantados. A toxoplasmose congênita pode provocar aborto ou até sérios danos ao feto provocando retardo mental e cegueira em crianças, reduzindo significativamente a qualidade de vida dos sobreviventes. Assim, a transmissão congênita pode ser muito mais importante do que se pensava. Estudos sobre a evolução da infecção por Toxoplasma gondii em diferentes tipos de células hospedeiras se fazem necessários para uma abordagem terapêutica adequada. Nesse estudo utilizamos as técnicas de imunofluorescência e imunocitoquímica ultraestrutural com o objetivo de investigar a participação de Bax e Bcl-2, proteínas da família Bcl-2 que regulam a apoptose e a dinâmica mitocondrial durante a evolução da infecção das células endoteliais de veia umbilical humana (HUVEC) por taquizoítos de T. gondii. A microscopia confocal revelou uma rede mitocondrial filamentosa marcada pelo Mito Tracker Red no citoplasma de HUVEC e após 2h de infecção essa rede se mostrou desorganizada, provavelmente por conseqüência da invasão da HUVEC pelo T. gondii. Porém, em 6h de infecção, observamos a reestruturação da rede mitocondrial no citoplasma de HUVEC que se manteve no tempo de 20h de infecção. Ainda por microscopia confocal observamos que a proteína pró-apoptótica Bax se localiza principalmente no citoplasma, na mitocôndria, e pela primeira vez foi detectada no núcleo de HUVEC. No tempo de 2h de infecção, observamos a expressão de Bax principalmente na mitocôndria. Entretanto, após 6h e 20h de infecção, essa expressão diminuiu tanto na mitocôndria quanto no citoplasma de HUVEC. A expressão de Bcl-2 não foi observada em HUVEC não infectada e infectada por 2h, 6h e 20h. Taquizoítos de T. gondii apresentaram marcação positiva para Bax e Bcl-2 ao longo de todos os tempos de infecção. A análise ultraestrutural confirmou a dinâmica mitocondrial observada por microscopia confocal durante toda a interação. Os resultados de imunocitoquímica confirmaram a expressão de Bax no núcleo de HUVEC e a expressão de Bax e Bcl-2 em taquizoítos de T. gondii isolados. Portanto, nossos resultados sugerem que o T. gondii modula a morfologia da mitocôndria e a expressão de Bax em HUVEC, interferindo possivelmente nos mecanismos de defesa das células hospedeiras, entre eles a progressão da apoptose. / Toxoplasmosis is a zoonosis widely distributed that affects over a third of world population and is of great importance on public health. Most Toxoplasma gondii infection in humans is asymptomatic. However, on recent years toxoplasmosis has been investigated since it comes as some opportunistic diseases in patients with the acquired immunodeficiency syndrome (AIDS) and transplant patients. Congenital toxoplasmosis can cause miscarriage or serious damage to the fetus leading to mental retardation and blindness in children, significantly reducing the quality of life of survivors. Thus, congenital transmission can be much more important than previously thought. Studies on the development of Toxoplasma gondii in different host cells are necessary for an appropriate therapy. At this study we employed the immunofluorescence and ultrastructural immunocytochemical assay with the aim to investigate the involvement of Bax and Bcl-2, members of Bcl-2 family which regulate apoptosis and the mitochondrial dynamic during the infection progress of human umbilical vein endothelial cells (HUVEC) by tachyzoites of T. gondii. Confocal microscopy revealed a mitochondrial filamentous network stained by Mito Tracker Red within cytoplasm of HUVEC and after 2h of infection this network became desorganized, probably as a consequence of T. gondii invasion. However, at 6h of infection, we observed the restructuring of the mitochondrial network in the cytoplasm of HUVEC, which remained at the time of 20h of infection. Even by confocal microscopy we observed Bax pro-apoptotic protein located mainly in the cytoplasm, in the mitochondria, and for the first time it was detected in the nucleus of HUVEC. At the time of 2h of infection, we observed the expression of Bax mainly in mitochondria. However, after 6h and 20h of infection, this expression decreased such in mitochondria as in HUVEC cytoplasm. The Bcl-2 expression was not observed in uninfected and HUVEC infected for 2h, 6h and 20h. Tachyzoites of T. gondii presented positive labeling for Bax and Bcl-2 over all time of infection. The ultrastructural analysis confirmed the mitochondrial dynamics observed by confocal microscopy throughout the interaction. The results of immunocytochemistry confirmed Bax expression in the nucleus of HUVEC and the expression of Bax and Bcl-2 in isolated tachyzoites of T. gondii. Therefore, our results suggest that T. gondii modulates the mitochondria morphology and Bax expression in HUVEC, probably interfering on the host cells defense mechanisms, including the progression of apoptosis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A toxoplasmose é uma zoonose amplamente distribuída que afeta mais de um terço da população mundial e de grande importância na saúde pública. A maioria das infecções em humanos por Toxoplasma gondii é assintomática. A toxoplasmose é amplamente investigada visto que se apresenta como uma doença grave em pessoas imunodeprimidas (portadores da síndrome da imunodeficiência adquirida (SIDA), não tratados, indivíduos transplantados, paciente em tratamento quimioterápico ou em uso de drogas supressoras e gestantes). A toxoplasmose congênita frequentemente pode levar ao aborto espontâneo ou até mesmo resultar na formação de crianças com algum grau de atraso no desenvolvimento mental e/ou físicos, deste modo, a transmissão congênita pode ser muito mais importante do que se pensava, pois os parasitos encontrados na circulação sanguinea são capazes de infectar as células endoteliais dos vasos e os tecidos circunjacentes, podendo resultar no encistamento do T. gondii. Atualmente a toxoplasmose vem sendo investigada devido a sua associação a inúmeras outras doenças, assim, estudos sobre a evolução da infecção por T. gondii em diferentes tipos de células hospedeiras se fazem necessários para uma abordagem terapêutica adequada. Ao invadir a célula hospedeira o parasito possui a capacidade de recrutar as mitocôndrias promovendo mudanças na organização mitocondrial ao longo da progressão da infecção, garantindo um ambiente favorável a sua multiplicação. Diante disso, investigamos se o parasito possui a capacidade de interferir no metabolismo mitocondrial e na resposta apoptótica da célula endotelial. O presente trabalho teve como objetivo analisar o metabolismo mitocondrial através da respirometria de alta-resolução e da resposta apoptótica através do western blotting das células endoteliais da veia umbilical humana (HUVEC) infectadas por 2, 6 e 20 horas por taquizoítos de T. gondii. A respirometria de alta-resolução revelou que o parasito interfere no metabolismo energético da célula hospedeira. A análise do conteúdo de proteínas da família Bcl-2 por western blotting revelou maior estímulo apoptótico no tempo inicial de infecção, quando comparado aos demais tempos. Os resultados dos conteúdos de caspase 3, proteína efetora da apoptose, não demonstrou diferença nos tempos iniciais de infecção Entretanto, em tempos mais tardios, o conteúdo de caspase 3 mostrou-se significativamente aumentado quando comparado às HUVEC não infectadas. A dinâmica de replicação do parasito foi observada através do monitoramento pelo sistema Time-Lapse Nikon BioStation IMQ em tempo real das células infectadas por T.gondii. Portanto, nossos resultados sugerem que o protozoário ao recrutar as mitocôndrias da célula hospedeira interfere no metabolismo mitocondrial e na modulação da apoptose para garantir um ambiente favorável a sua multiplicação. / Toxoplasmosis is a widespread zoonosis that affects more than a third of the world population and of great public health importance. Most human infections with Toxoplasma gondii are asymptomatic. Toxoplasmosis is widely investigated since it presents itself as a serious disease in immunocompromised persons (holders of acquired immunodeficiency syndrome (AIDS), untreated, transplant recipients, patients undergoing chemotherapy or suppressing drugs and pregnant). Congenital toxoplasmosis can often lead to miscarriage or even result in the formation of children with some degree of developmental delay mental and / or physical, thus congenital transmission may be much more important than previously thought, because the parasites found In the bloodstream are able to infect endothelial cells of blood vessels and surrounding tissues, which may result in encystment T. gondii. Currently toxoplasmosis has been investigated because of their association with other diseases, so, studies of the evolution of T.gondii infection in different types of host cells are necessary for an adequate therapeutic approach. To invade the host cell, the parasite has the ability to recruit mitochondria promoting changes in mitochondrial organization along the progression of infection, ensuring a favorable environment for their multiplication. Therefore, we investigated whether the parasite has the ability to interfere with mitochondrial metabolism and apoptotic response of endothelial cells. This study aimed to analyze the mitochondrial metabolism by high-resolution respirometry and apoptotic response by western blotting of endothelial cells of human umbilical vein (HUVEC) infected for 2, 6 and 20 hours per tachyzoites of T. gondii. The high-resolution respirometry revealed that the parasite interferes with the energy metabolism of the host cell. The analysis of the family protein content of Bcl-2 by western blotting revealed higher apoptotic stimulus at the initial time of infection, as compared to other times. The results of the contents of caspase 3 protein effector of apoptosis, showed no difference in the initial days of infection However, in more recent times, the content of caspase 3 was significantly increased when compared to non-infected HUVEC. The dynamics of parasite replication was observed by monitoring the system Time-Lapse Nikon BioStation IMQ in real time from infected cells by T. gondii. Therefore, our findings suggest that mitochondria in recruiting protozoan host cell interfere with mitochondrial metabolism and in the modulation of apoptosis to ensure a favorable environment for multiplication.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A toxoplasmose é uma zoonose amplamente distribuída que afeta mais de um terço da população mundial e de grande importância na saúde pública. A maioria das infecções em humanos por Toxoplasma gondii é assintomática. A toxoplasmose é amplamente investigada visto que se apresenta como uma doença grave em pessoas imunodeprimidas (portadores da síndrome da imunodeficiência adquirida (SIDA), não tratados, indivíduos transplantados, paciente em tratamento quimioterápico ou em uso de drogas supressoras e gestantes). A toxoplasmose congênita frequentemente pode levar ao aborto espontâneo ou até mesmo resultar na formação de crianças com algum grau de atraso no desenvolvimento mental e/ou físicos, deste modo, a transmissão congênita pode ser muito mais importante do que se pensava, pois os parasitos encontrados na circulação sanguinea são capazes de infectar as células endoteliais dos vasos e os tecidos circunjacentes, podendo resultar no encistamento do T. gondii. Atualmente a toxoplasmose vem sendo investigada devido a sua associação a inúmeras outras doenças, assim, estudos sobre a evolução da infecção por T. gondii em diferentes tipos de células hospedeiras se fazem necessários para uma abordagem terapêutica adequada. Ao invadir a célula hospedeira o parasito possui a capacidade de recrutar as mitocôndrias promovendo mudanças na organização mitocondrial ao longo da progressão da infecção, garantindo um ambiente favorável a sua multiplicação. Diante disso, investigamos se o parasito possui a capacidade de interferir no metabolismo mitocondrial e na resposta apoptótica da célula endotelial. O presente trabalho teve como objetivo analisar o metabolismo mitocondrial através da respirometria de alta-resolução e da resposta apoptótica através do western blotting das células endoteliais da veia umbilical humana (HUVEC) infectadas por 2, 6 e 20 horas por taquizoítos de T. gondii. A respirometria de alta-resolução revelou que o parasito interfere no metabolismo energético da célula hospedeira. A análise do conteúdo de proteínas da família Bcl-2 por western blotting revelou maior estímulo apoptótico no tempo inicial de infecção, quando comparado aos demais tempos. Os resultados dos conteúdos de caspase 3, proteína efetora da apoptose, não demonstrou diferença nos tempos iniciais de infecção Entretanto, em tempos mais tardios, o conteúdo de caspase 3 mostrou-se significativamente aumentado quando comparado às HUVEC não infectadas. A dinâmica de replicação do parasito foi observada através do monitoramento pelo sistema Time-Lapse Nikon BioStation IMQ em tempo real das células infectadas por T.gondii. Portanto, nossos resultados sugerem que o protozoário ao recrutar as mitocôndrias da célula hospedeira interfere no metabolismo mitocondrial e na modulação da apoptose para garantir um ambiente favorável a sua multiplicação. / Toxoplasmosis is a widespread zoonosis that affects more than a third of the world population and of great public health importance. Most human infections with Toxoplasma gondii are asymptomatic. Toxoplasmosis is widely investigated since it presents itself as a serious disease in immunocompromised persons (holders of acquired immunodeficiency syndrome (AIDS), untreated, transplant recipients, patients undergoing chemotherapy or suppressing drugs and pregnant). Congenital toxoplasmosis can often lead to miscarriage or even result in the formation of children with some degree of developmental delay mental and / or physical, thus congenital transmission may be much more important than previously thought, because the parasites found In the bloodstream are able to infect endothelial cells of blood vessels and surrounding tissues, which may result in encystment T. gondii. Currently toxoplasmosis has been investigated because of their association with other diseases, so, studies of the evolution of T.gondii infection in different types of host cells are necessary for an adequate therapeutic approach. To invade the host cell, the parasite has the ability to recruit mitochondria promoting changes in mitochondrial organization along the progression of infection, ensuring a favorable environment for their multiplication. Therefore, we investigated whether the parasite has the ability to interfere with mitochondrial metabolism and apoptotic response of endothelial cells. This study aimed to analyze the mitochondrial metabolism by high-resolution respirometry and apoptotic response by western blotting of endothelial cells of human umbilical vein (HUVEC) infected for 2, 6 and 20 hours per tachyzoites of T. gondii. The high-resolution respirometry revealed that the parasite interferes with the energy metabolism of the host cell. The analysis of the family protein content of Bcl-2 by western blotting revealed higher apoptotic stimulus at the initial time of infection, as compared to other times. The results of the contents of caspase 3 protein effector of apoptosis, showed no difference in the initial days of infection However, in more recent times, the content of caspase 3 was significantly increased when compared to non-infected HUVEC. The dynamics of parasite replication was observed by monitoring the system Time-Lapse Nikon BioStation IMQ in real time from infected cells by T. gondii. Therefore, our findings suggest that mitochondria in recruiting protozoan host cell interfere with mitochondrial metabolism and in the modulation of apoptosis to ensure a favorable environment for multiplication.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A toxoplasmose é uma zoonose amplamente distribuída que afeta mais de um terço da população mundial e de grande importância na saúde pública. A maioria das infecções em humanos por Toxoplasma gondii é assintomática. Entretanto, nos últimos anos, a toxoplasmose tem sido amplamente investigada uma vez que se apresenta como uma das doenças oportunistas que acometem pacientes portadores da Síndrome da Imunodeficiência Adquirida (AIDS) e indivíduos transplantados. A toxoplasmose congênita pode provocar aborto ou até sérios danos ao feto provocando retardo mental e cegueira em crianças, reduzindo significativamente a qualidade de vida dos sobreviventes. Assim, a transmissão congênita pode ser muito mais importante do que se pensava. Estudos sobre a evolução da infecção por Toxoplasma gondii em diferentes tipos de células hospedeiras se fazem necessários para uma abordagem terapêutica adequada. Nesse estudo utilizamos as técnicas de imunofluorescência e imunocitoquímica ultraestrutural com o objetivo de investigar a participação de Bax e Bcl-2, proteínas da família Bcl-2 que regulam a apoptose e a dinâmica mitocondrial durante a evolução da infecção das células endoteliais de veia umbilical humana (HUVEC) por taquizoítos de T. gondii. A microscopia confocal revelou uma rede mitocondrial filamentosa marcada pelo Mito Tracker Red no citoplasma de HUVEC e após 2h de infecção essa rede se mostrou desorganizada, provavelmente por conseqüência da invasão da HUVEC pelo T. gondii. Porém, em 6h de infecção, observamos a reestruturação da rede mitocondrial no citoplasma de HUVEC que se manteve no tempo de 20h de infecção. Ainda por microscopia confocal observamos que a proteína pró-apoptótica Bax se localiza principalmente no citoplasma, na mitocôndria, e pela primeira vez foi detectada no núcleo de HUVEC. No tempo de 2h de infecção, observamos a expressão de Bax principalmente na mitocôndria. Entretanto, após 6h e 20h de infecção, essa expressão diminuiu tanto na mitocôndria quanto no citoplasma de HUVEC. A expressão de Bcl-2 não foi observada em HUVEC não infectada e infectada por 2h, 6h e 20h. Taquizoítos de T. gondii apresentaram marcação positiva para Bax e Bcl-2 ao longo de todos os tempos de infecção. A análise ultraestrutural confirmou a dinâmica mitocondrial observada por microscopia confocal durante toda a interação. Os resultados de imunocitoquímica confirmaram a expressão de Bax no núcleo de HUVEC e a expressão de Bax e Bcl-2 em taquizoítos de T. gondii isolados. Portanto, nossos resultados sugerem que o T. gondii modula a morfologia da mitocôndria e a expressão de Bax em HUVEC, interferindo possivelmente nos mecanismos de defesa das células hospedeiras, entre eles a progressão da apoptose. / Toxoplasmosis is a zoonosis widely distributed that affects over a third of world population and is of great importance on public health. Most Toxoplasma gondii infection in humans is asymptomatic. However, on recent years toxoplasmosis has been investigated since it comes as some opportunistic diseases in patients with the acquired immunodeficiency syndrome (AIDS) and transplant patients. Congenital toxoplasmosis can cause miscarriage or serious damage to the fetus leading to mental retardation and blindness in children, significantly reducing the quality of life of survivors. Thus, congenital transmission can be much more important than previously thought. Studies on the development of Toxoplasma gondii in different host cells are necessary for an appropriate therapy. At this study we employed the immunofluorescence and ultrastructural immunocytochemical assay with the aim to investigate the involvement of Bax and Bcl-2, members of Bcl-2 family which regulate apoptosis and the mitochondrial dynamic during the infection progress of human umbilical vein endothelial cells (HUVEC) by tachyzoites of T. gondii. Confocal microscopy revealed a mitochondrial filamentous network stained by Mito Tracker Red within cytoplasm of HUVEC and after 2h of infection this network became desorganized, probably as a consequence of T. gondii invasion. However, at 6h of infection, we observed the restructuring of the mitochondrial network in the cytoplasm of HUVEC, which remained at the time of 20h of infection. Even by confocal microscopy we observed Bax pro-apoptotic protein located mainly in the cytoplasm, in the mitochondria, and for the first time it was detected in the nucleus of HUVEC. At the time of 2h of infection, we observed the expression of Bax mainly in mitochondria. However, after 6h and 20h of infection, this expression decreased such in mitochondria as in HUVEC cytoplasm. The Bcl-2 expression was not observed in uninfected and HUVEC infected for 2h, 6h and 20h. Tachyzoites of T. gondii presented positive labeling for Bax and Bcl-2 over all time of infection. The ultrastructural analysis confirmed the mitochondrial dynamics observed by confocal microscopy throughout the interaction. The results of immunocytochemistry confirmed Bax expression in the nucleus of HUVEC and the expression of Bax and Bcl-2 in isolated tachyzoites of T. gondii. Therefore, our results suggest that T. gondii modulates the mitochondria morphology and Bax expression in HUVEC, probably interfering on the host cells defense mechanisms, including the progression of apoptosis.

L'apoptose est un processus cellulaire fondamental pour l'homéostasie tissulaire. Ce type de mort cellulaire est sous le contrôle des protéines de la famille Bcl-2 qui régulent la perméabilité de la membrane externe de la mitochondrie. Cependant, au-delà de leur rôle dans le contrôle de l'apoptose, les protéines de la famille Bcl-2 peuvent intervenir dans d'autres processus tels que le cycle cellulaire ou le métabolisme. Au sein du laboratoire, nous nous intéressons tout particulièrement aux rôles non-apoptotiques des protéines Bcl-2 au cours du développement embryonnaire. Grâce à l'utilisation du poisson zèbre, nous avons pu montrer que les protéines de la famille Bcl-2 contrôlent différents processus au cours du développement grâce à leur capacité à réguler l'homéostasie calcique. En effet, nous avons montré que la protéine anti-apoptotique Nrz participe au remodelage du cytosquelette d'actine au cours de l'épibolie en régulant la concentration de calcium cytosolique par son interaction avec le récepteur à l'IP3 (IP3R). Nous avons de plus pu montrer que Nrz diminue la sortie de calcium du réticulum endoplasmique en inhibant la fixation de l'IP3 sur son récepteur. Nous avons également identifié un nouveau membre pro-apoptotique de la famille Bcl-2, Bclwav, spécifiquement exprimée chez les poissons et le xénope. Cette protéine participe à la régulation de l'homéostasie calcique mitochondriale en interagissant avec VDAC. Nous avons de plus montré que cette activité est essentielle pour les mouvements de convergence et d'extension au cours du développement embryonnaire précoce du poisson zèbre / Apoptosis is a key cellular process for tissue homeostasis. Apoptotic cell death is under control of Bcl-2 family proteins which regulate outer mitochondrial membrane permeability. However, beyond their role in apoptosis, Bcl-2 family proteins are also involved in other cellular processes such as cell cycle or metabolism. In our laboratory we are interested in non-apoptotic functions of Bcl-2 family proteins in embryonic development. Using zebrafish model we have shown that Bcl-2 proteins control different processes during early development thanks to their ability to regulate calcium homeostasis. Indeed, we have shown that the anti-apoptotic protein Nrz participates in actin cytoskeleton remodeling during epiboly by regulating cytosolic calcium concentration via an interaction with the IP3 receptor (IP3R). We have also demonstrated that Nrz decreases calcium release from the endoplasmic reticulum by inhibiting IP3 fixation on its receptor. We have furthermore identified a new pro-apoptotic member of Bcl-2 family, Bcl-wav which is expressed only in fish and frogs. This protein regulates mitochondrial calcium homeostasis by interacting with VDAC. We have moreover shown that this activity is essential for convergence and extension movements during early zebrafish development

O splicing alternativo do pré-mRNA de BCL-X produz duas isoformas de mRNAs com funções antagônicas, a pró-apoptótica BCL-XS e a anti-apoptótica BCL-XL, cujo balanço regula a homeostasia celular. Entretanto, o mecanismo que regula esse processamento ainda é desconhecido. Nesse trabalho, nós identificamos e caracterizamos um longo RNA não codificador de proteínas (lncRNA) nomeado INXS, que é transcrito a partir da fita oposta do locus genômico de BCL-X, sendo menos abundante em linhagens celulares tumorais e tecidos tumorais de pacientes quando comparados com os respectivos pares não tumorais. INXS é um RNA unspliced de 1903 nts, é transcrito pela RNA Polimerase II, possui cap 5\', está enriquecido na fração nuclear das células e se liga à proteína Sam68 do complexo modulador de splicing. O tratamento de células tumorais 786-O com cada um de três agentes indutores de apoptose aumentou a expressão endógena do INXS, levando ao aumento expressivo da proporção entre os mRNAs de BCL-XS / BCL-XL, e ativação das caspases 3, 7 e 9. Estes efeitos foram anulados na presença do knockdown do INXS. Da mesma forma, a superexpressão ectópica do INXS causou uma mudança no splicing favorecendo a isoforma BCL-XS e ativação das caspases, aumentando os níveis da proteína BCL-XS e conduzindo as células à apoptose. Utilizando um modelo in vivo, cinco injeções intra-tumorais do INXS durante 15 dias causaram uma regressão acentuada no volume dos xenotumores. Portanto, INXS é um lncRNA que induz a apoptose, sugerindo que essa molécula seja um possível alvo a ser explorado na terapia contra o câncer. / BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL, whose balance regulates cell homeostasis. However, the mechanism that regulates the splice shifting is incompletely understood. Here, we identified and characterized a long noncoding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was less abundant in tumor cell lines and patient tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA Polymerase II, 5\'-capped, nuclear enriched and binds Sam68 splicing-modulator. The treatment of tumor cell line 786-O with each of three apoptosis-inducing agents increased endogenous INXS lncRNA, increased BCL-XS / BCL-XL mRNA ratio, and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing towards BCL-XS and activation of caspases, increasing the levels of BCL-XS protein and then leading the cells to apoptosis. In a mouse xenograft model, five intra-tumor injections of INXS along 15 days caused a marked regression in tumor volume. INXS is an lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

Notre équipe étudie les mécanismes de résistance à l’apoptose induite par divers agents dans les cellules de lymphomes B infectées ou non par le virus d’Epstein-Barr (EBV). EBV est un virus oncogénique de la famille des gamma-herpès virus qui est associé notamment au lymphome de Burkitt (LB) et aux syndromes lymphoprolifératifs post-transplantation (PTLD). Des résultats précédents ont montré que l’utilisation de la nutline-3, une molécule capable de se fixer sur MDM2, active p53 dans ces cellules tumorales. Cependant cette activation de p53 provoque l’apoptose des cellules B EBV(-) alors que les cellules B EBV(+) en latence III (exprimant toutes les protéines virales dites « de latence ») sont beaucoup plus résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans cette résistance afin de mettre en place des stratégies thérapeutiques pour la contourner. La première partie de ma thèse a été consacrée à l’étude du rôle de l’autophagie dans la résistance des cellules EBV(+) en latence III à l’apoptose. L’autophagie est un processus de dégradation des protéines qui joue un rôle physiologique complexe impliqué à la fois dans la survie et dans la mort cellulaire. Les travaux effectués ont montré que: 1) l’autophagie est induite en réponse au traitement par la nutline dans les cellules EBV(+) en latence III ; 2) ces cellules expriment fortement la Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie participe à la résistance de ces cellules à l’apoptose. La seconde partie de ma thèse a été consacrée au développement de nouvelles molécules ciblant les protéines anti-apoptotiques de la famille de Bcl-2. En effet, outre Bcl-2 qui est surexprimé dans les cellules EBV(+), les cellules de LB et les PTLD surexpriment aussi Mcl-1, une autre protéine anti-apoptotique. Or il a été montré que cette protéine était fréquemment à l’origine de résistance à des inhibiteurs déjà développés (et en essais cliniques) contre Bcl-2. Le développement de molécules ciblant Mcl-1 s’avère donc utile pour les contrer. Pour cela une collaboration avec une équipe de chimiste (dirigée par Fanny Roussi à l’Institut de Chimie des Substances Naturelles à Gif-sur-Yvette) a été mise en place. Nous avons identifié et étudié les mécanismes d’action de plusieurs molécules inhibitrices potentielles de Mcl-1 et/ou Bcl-xL capables d’induire l’apoptose dans nos deux modèles de lymphomes. / Our team investigates the mechanisms of resistance to apoptosis induced in various B-cell lymphomas including some infected by the Epstein-Barr virus (EBV). EBV is an oncogenic member in the gamma-herpesvirus family. Among other pathologies, it is associated with Burkitt’s lymphoma (BL) and post-transplant lymphoproliferative disorders (PTLD). Previously, our laboratory has found that in these tumor cells, the binding of nutlin-3 to MDM2 results in the activation of p53. However, although p53 activation leads to apoptosis in EBV(-) cells, EBV(+) latency III cells which express all viral « latency » proteins are much more resistant. During this PhD project, I studied the mechanisms involved in this resistance and made attempts to define new therapeutic strategies that would bypass them. First, the role played by autophagy was investigated. This catabolic process which degrades proteins and organelles is physiologically complex as it is involved in both cell survival and cell death. Our work has demonstrated that: 1) autophagy was induced in nutlin-3 treated EBV(+) latency III cells; 2) Beclin-1 was strongly expressed in these cells whose autophagy was constitutively activated; 3) autophagy was involved in the resistance to apoptosis observed in these cells. Second, I turned my efforts to the identification of new molecules targeting anti-apoptotic members of the Bcl-2 family. Like Bcl-2, the antiapoptotic protein Mcl-1 is heavily expressed in LB and PTLD cell lines but in this case, independently of their EBV status and this is a frequent cause for the observed resistance to Bcl-2 inhibitors that are currently tested in clinical trials. Molecules targeting Mcl-1 could thus prove promising to circumvent this resistance. In a collaboration with a Chemistry team supervised by Fanny Roussi at the Institut de Chimie des Substances Naturelles in Gif-sur-Yvette, we have identified the mechanisms of action of potential inhibitors of Mcl-1 and/or Bcl-xL, another anti-apoptotic molecules which induce apoptosis in our two lymphoma models.

L'apoptose, appelée aussi mort cellulaire programmée, est un processus physiologique qui permet d'éliminer les cellules endommagées ou superflues lors du développement cellulaire. Le phénomène d'échappement à l'apoptose est une caractéristique fondamentale d'une cellule cancéreuse. Une des voies d'induction principale de l'apoptose est contrôlée par la famille des protéines Bcl-2 qui comprend des protéines pro-apoptotiques (comme Bak ou Bid) et des protéines anti-apoptotiques (comme Bcl-xL, Bcl-2 et Mcl-1). Dans de nombreux cancers, certaines protéines anti-apoptotiques de cette famille sont surexprimées ce qui induit une inhibition de l'apoptose et une résistance aux traitements classiques. Cette famille de protéines est une cible stratégique et prometteuse pour développer une nouvelle classe d'agents anticancéreux. Notre équipe a entrepris un criblage d'extraits bruts de plantes d'origine géographique variée sur l'interaction Bcl-xL/Bak. Cette étude préliminaire a conduit à la caractérisation de la meiogynine A, un sesquiterpène dimère isolé des écorces de Meiogyne cylindrocarpa, une plante de Malaisie. Ce composé est un inhibiteur naturel de l'interaction entre Bcl-xL et le peptide Bak et entre Mcl-1 et le peptide Bid. La synthèse totale de la meiogynine A a été développée dans l'équipe et repose sur une cycloaddition de Diels-Alder entre un triène et un diénophile. Deux analogues de 1ère génération, où le cyclohexane a été remplacé par un cycle aromatique, ont été synthétisés et ont montré une bonne activité in vitro et in cellulo. Cependant, la cycloaddition de Diels-Alder est très longue. Dans un premier temps, afin d'améliorer la réactivité des triènes cibles et d'effectuer des relations structure activité au niveau de la partie "Sud" de la meiogynine A, la synthèse de triènes chlorés originaux diversement fonctionnalisés au niveau de la partie aromatique a été réalisée. Deux diénophiles différents ont également été synthétisés en vue de modifier la partie "Est" de la meiogynine A. Plusieurs analogues ont ainsi été obtenus et ont été évalués biologiquement sur les protéines anti-apoptotiques Bcl-xL et Mcl-1 sur des tests in vitro. Dans un deuxième temps, des études préliminaires de l'interaction protéine/ligand ont été commencées en synthétisant deux sondes photoactivables. / Apoptosis, or programmed death, is used by multicellular organisms to regulate tissue homeostasis through the elimination of useless or potentially harmful cells. One of the main apoptotic pathways is controlled by the Bcl-2 family of proteins. These proteins are divided into pro-apoptotic members (as Bak or Bid) and anti-apoptotic members (as Bcl-xL, Mcl-1 or Bcl-2). In some cancers, they are often overexpressed in many kinds of cancer or are involved in the resistance to chemotherapy. Targeting these proteins is a highly promising strategy for anticancer therapy that has emerged over the last decades. Our team underwent a bioassay-guided screening of various plants extracts. A few years ago, meiogynin A, a dimeric sesquiterpenoid was isolated from a Malaysian tree bark using a bioassay-guided screening. This compound is a natural bcl-xL and Mcl-1 inhibitor. Total synthesis of meiogynine A was developped by our team and the final step is a Diels-Alder cycloaddition reaction between a triene and a dienophile. Then, the synthesis of two anlogues was performed, where the cyclohexane is replaced by an aromatic, and they shown a good biological activity in vitro and in celullo. Nevertheless, the Diels-Alder reaction is very slow. In order to improve triene reactivity and to perform structure activity relationship in the south part of meiogynine A, the synthesis of various original chlorinated triene functionnalized in the aromatic was realised. Two differents dienophiles was synthesized in order to modify the east part of the molecule. Several analogues were obtained et were evaluated on Bcl-xL and Mcl-1 proteins.Also, preliminary studies on protein/ligand interaction was started by the synthesis of two photoactivable probes.

Les protéines BCL-2 jouent un rôle essentiel dans la décision de vie ou de mort des cellules. Elles contrôlent l'induction de l'apoptose (mort cellulaire programmée) par la voie mitochondriale via des fonctions opposées de régulateurs anti- et pro-apoptotiques. Les protéines contenant un ou plusieurs domaines dits d'homologie à Bcl-2 (BHl- 4) sont systématiquement classées dans cette famille. Grâce à une analyse bioinformatique et phylogénétique, nous avons revisité les différents critères d'inclusion dans le groupe de protéines BCL-2 et proposé une nouvelle classification tenant compte des données structurales et évolutives. Cette nouvelle nomenclature distingue : un premier groupe de protéines homologues (dérivant d'un ancêtre commun), partageant une structure 3D semblable à celle de Bcl-2 et pouvant ne posséder aucun motif BH, et un conglomérat, en pleine expansion, regroupant des protéines sans lien phylogénétique apparent et partageant une courte région de similarité de séquence correspondant au motif BH3. Sur la base de ces résultats, nous avons construit un processus, basé sur des profils HMM, pour identifier les protéines appartenant au groupe de protéines BCL-2. Notre processus automatisé est utilisé pour i) récupérer les séquences nucléotidiques et protéiques mensuellement ii) les annoter et iii) les intégrer dans la base de connaissances BCL2DB (« The BCL-2 Database »). Celle-ci est accessible via une interface Web (http://bcl2db.ibcp.fr) qui permet aux chercheurs d'extraire des données et d'effectuer des analyses de séquence / BCL-2 proteins play an essential role in the decision of life or death of animal cells. They control the induction of apoptosis (programmed cell death) in the mitochondrial pathway via regulators having opposite functions: anti- or pro-apoptotic. Proteins containing one or more Bcl-2 homology domains (BHl-4) are systematically classified in this family. Through bioinformatics and phylogenetic analysis, we revisited the different criteria for protein inclusion in the BCL-2 group and proposed a new classification taking into account structural and evolutionary data. This new nomenclature distinguishes a first group of homologous proteins (derived from a common ancestor), sharing a similar 3D structural fold with Bcl-2 and often (but not necessarily) having one or more BH motifs, and a fast expanding conglomerate of proteins without apparent phylogenetic relationships and sharing only a short region of sequence similarity corresponding to the BH3 motif. Based on these results, we built a process based on profiles HMM to identify proteins belonging to the BCL-2 protein group. Our automated process i) recovers on a monthly basis the nucleotide and protein sequences ii) annotates them and iii) integrates this information into BCL2DB ("The BCL-2 Database"). This resource can be accessed via a web interface (http://bcl2db.ibcp.fr) which allows researchers to extract data and perform sequence analysis